A recoding element that stimulates decoding of UGA codons by Sec tRNA^[Ser]Sec

Abstract

Selenocysteine insertion during decoding of eukaryotic selenoprotein mRNA requires several trans-acting factors and a cis-acting selenocysteine insertion sequence (SECIS) usually located in the 39 UTR. A second cis-acting selenocysteine codon redefinition element (SRE) has recently been described that resides near the UGA-Sec codon of selenoprotein N (SEPN1). Similar phylogenetically conserved elements can be predicted in a subset of eukaryotic selenoprotein mRNAs. Previous experimental analysis of the SEPN1 SRE revealed it to have a stimulatory effect on readthrough of the UGA-Sec codon, which was not dependent upon the presence of a SECIS element in the 39 UTR; although, as expected, readthrough efficiency was further elevated by inclusion of a SECIS. In order to examine the nature of the redefinition event stimulated by the SEPN1 SRE, we have modified an experimentally tractable in vitro translation system that recapitulates efficient selenocysteine insertion. The results presented here illustrate that the SRE element has a stimulatory effect on decoding of the UGA-Sec codon by both the methylated and unmethylated isoforms of Sec tRNA [Ser]Sec , and confirm that efficient selenocysteine insertion is dependent on the presence of a 39-UTR SECIS. The variation in recoding elements predicted near UGA-Sec codons implies that these elements may play a differential role in determining the amount of selenoprotein produced by acting as controllers of UGA decoding efficiency.

Functional Analysis of the Interplay between Translation Termination, Selenocysteine Codon Context, and Selenocysteine Insertion Sequence-binding Protein 2

“…tein N, which, in addition to the SECIS element, has a cis-element in its coding region that stimulates Sec incorporation (26). Juxtaposing the previous findings to those in this paper, it appears likely that in selenoproteins, a larger codon context in conjunction with the base downstream of the UGA codon, determine readthrough.…”

Characterization of the UGA-recoding and SECIS-binding activities of SECIS-binding protein 2

“…33,34 As shown in Figure 1B, maximal luciferase activity was obtained using 80 nM purified bacterially expressed SBP2-CT, which is comparable to reports in the literature that range from 40-300 nM. [35][36][37][38][39] In contrast, 0.4 nM in vitro translated SBP2-CT generates an equivalent signal to 40 nM of recombinant protein (compare Figs. 1B and C), suggesting that the in vitro translated protein is significantly more active than the bacterially expressed protein.…”

Modeling and gene knockdown to assess the contribution of nonsense-mediated decay, premature termination, and selenocysteine insertion to the selenoprotein hierarchy

“…Another proposed mechanism is the use of two different tRNAsec isoforms in selenocysteine insertion by selenoprotein mRNAs and a change in expression of one of the isoforms when Se supply is low (Howard et al 2007(Howard et al , 2013. Although an effect of the tRNAsec isoforms has been demonstrated, it was rather small, and it is not clear how it could be used to explain the rather large change in mRNA abundance observed at low Se concentrations.…”